Cholesteryl ester is the major lipid species accumulated largely as cytosolic droplets in human atherosclerotic plaques. The formation of intracellular cholesteryl esters is catalyzed by the enzyme acyl coenzyme A: cholesterol acyltransferase (ACAT). ACAT is an integral membrane protein present in minute quantity in the endoplasmic reticulum. The structure of ACAT, as well as the molecular mechanism(s) for ACAT regulation is not understood at present. We propose.to produce and characterize various specific molecular probes for the enzyme ACAT, including the cDNA and monospecific antibodies. We will next use these probes as tools to study the biochemical properties and the regulation of ACAT in cell culture as well as in animals. Our aims are: 1. To clone the human ACAT (H-ACAT) gene, and to sequence its fulllength cDNA. 2. To select and characterize stable cell lines overproducing the H-ACAT enzyme due to amplification of the H-ACAT gene. 3. To raise polyclonal and antipeptide antibodies against H-ACAT protein. 4. To study the topography of H-ACAT in sealed microsomal vesicles, and in reconstituted liposomes. 5. To examine phospholipid specificity of ACAT activity in reconstituted liposomes. 6. To quantitate the ACAT protein content, to study the rate of protein synthesis and degradation of ACAT, and to examine the potential enzymatic phosphorylation-dephosphorylation mechanism for regulating ACAT in intact cells as well as in rat liver. 7. To study the distribution of ACAT mRNA transcripts in mouse tissues, and to produce and characterize transgenic mice expressing H-ACAT gene. 8. To study the effects of anti-sense RNA and anti-sense oligonucleotide of H-ACAT cDNA on ACAT activity in cultured cells. 9. In vitro mutagenesis of H-ACAT cDNA, and functional analyses of the mutants. 10. Further characterization of cholesterol trafficking (CT) mutant.